The tetracyclines are bacteriostatic antibiotics used to treat a broad spectrum of microbial disease agents in humans, animals and plants.
Many bacteria are able to adapt to their environment in ways which permit them to become resistant to antibiotics. Strains of group A streptococci devoloped resistance to sulfadiazine during World War II. Resistant staphylococcal infections began to spread through public institutions and hospitals following the widespread use of penicillin. Since the introduction of tetracyclines into clinical practice, a number of microorganisms have developed resistance to these drugs. Treatment of bacterial infections in palm trees with tetracycline solutions through the root system has become increasingly ineffective. See Levy, "The Tetracyclines: Microbial Sensitivity and Resistance," New Trends in Antibiotics: Research and Therapy, Elsevier/North-Holland Biomedical Press, 1981, pp. 27-44; Chopra et al., "The tetracyclines: prospects at the beginning of the 1980's," Journal of Antimicrobial Chemotherapy, 8:5-21, (1981,) which are incorporated herein by reference.
Tetracycline was one of the real wonder drugs when introduced into the clinical world in 1948. However, many microorganisms have developed resistance to tetracycline. More alarming is the emergence of organisms with resistance to the newer tetracycline analogs. Resistant bacteria are also appearing among individuals who have not consciously ingested the drug. It has therefore become increasingly important to determine the mode of resistance and to develop a method of circumvention. Since the mechanism does not degrade the drug and breakdown in nature is small, tetracycline remains in the environment to continue to promote emergence of resistant organisms. Thus determination of a method that would overcome this resistance would provide a substantial increase in the effectiveness of tetracyclines, while reducing the present large increase in tetracycline resistant microbial diseases.
Investigations into the mode of action of the tetracyclines support observations that the tetracyclines inhibit the protein synthesis of sensitive bacteria at the level of the ribosome, as described in Lehninger, Biochemistry--The Molecular Basis of Cell Structure and Function, (2d ed., Worth Publishers, 1975), p. 941, which is incorporated herein by reference. This inhibition interferes with total protein synthesis and biosynthesis of the bacterial respiratory system.
In the resistant organism, resistance does not promote inactivation of the tetracycline molecule. Rather, the total efflux rate is increased and the steady state accumulation by the cell is obtained at a lower, biologically ineffective concentration of drug.
Resistance to the tetracyclines in most bacterial species is specified by extra-chromosomal, autonomously replicating and often transmissible plasmids, called R factors, which carry genes which mediate tetracycline resistance. Two kinds of tetracycline resistance determinants among plasmids have been described: those with resistance to tetracycline alone and those with resistance to tetracycline and its lipophilic analogs. It has been shown that at least four different genetic elements encode the tetracycline resistance phenotype. See Mendez et al., "Heterogeneity of Tetracycline Resistance Determinants," Plasmid, 3:99-108, 1980, which is incorporated herein by reference.
Plasmid mediated resistance is inducible in many bacteria. The resistance level can be experimentally increased by preincubation of the cells in subinhibitory amounts of tetracycline. It was found that coincident with induced resistance was the induced synthesis of a plasmid-encoded inner membrane protein, which was designated "TET" protein. See Levy and McMurry, "Detection of an Inducible Membrane Protein Associated with R-Factor-Mediated Tetracycline Resistance." Biochemical and Biophysical Research Communications, 56(4):1060-68, (1974), which is incorporated herein by reference. Moreover, accumulation of drug by resistant cells was dramatically different from that accumulation in sensitive cells. See Levy and McMurry, "Plasmid-determined Tetracycline Resistance involves new transport systems for tetracycline, " Nature, 275 (5683): 90-92 (1978), which is incorporated herein by reference. While tetracycline was actively accumulated by sensitive cells (McMurray and Levy in "Two transport systems for Tetracycline in Sensitive Escherichia coli: Critical role for an Initial Rapid Uptake System Insensitive to Energy Inhibitors," Antimicrobial Agents and Chemotherapy 14(2); 201-09 (1978), which is incorporated herein by reference), these uptake systems were found to be altered by at least one tetracycline resistance plasmid, R222. Nature 275 (5683), supra, at 91. They subsequently demonstrated that all four plasmid-borne tetracycline resistance determinants specified an active efflux system for tetracycline, (McMurray et al., "Active Efflux of Tetracycline Encoded by Four Genetically Different Tetracycline Resistance Determinants in Escherichia coli," Pro. Nat. Acad. of Sci. USA 77 (7): 3974-77 (1980), which is incorporated herein by reference). More recently, using tetracycline sensitive mutations which mapped in the TET structural region, the investigators demonstrated two genetic-complementation groups designated TET A and TET B. Absence of either one of these gene loci causes loss of the energy-dependent efllux of tetracycline which is characteristic of tetracycline resistance. (Curiale and Levy, "Two Complementation Groups Mediate Tetracycline Resistance Determined by Tn10," Journal of Bacteriology, 151(1): 209-15 (1982), which is incorporated herein by reference.)
It is among the objects of the present invention to provide a process for enhancing the bacteriostatic and bacteriocidal effects of the tetracyclines. It is also an object to provide a process for circumventing the tetracycline efflux mechanism of resistant bacterial cells. It is also an object to provide a process for promoting accumulation of minimum inhibitory concentrations of tetracyclines within the bacterial cell. It is also an object to provide a process for converting a tetracycline resistant cell into a tetracycline sensitive cell. Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out in the appended claims.